The objectives of this proposal are to identify and characterize novel proteins involved in mitochondrial iron/heme metabolism identified from differentiating fetal liver population by mRNA sequencing (RNAseq) and bioinformatics approaches. Heme serves as a prosthetic group in hemo-proteins for a wide array of crucial cellular processes, and of these, hemoglobin synthesis in red cells is the most well known. Despite advances in our understanding of cytosolic iron trafficking and proto-prophyrin biosynthesis, significant gaps remain, especially, with respect to components involving the egress of iron from the endosomes to the mitochondria, the trafficking of iron/heme within the mitochondria, and the eventual export of heme from the mitochondria for its incorporation in hemoglobin. In recent years, we have characterized the role ofthe l\/litoferrin1 (l\/lfrn1, Slc25a37) iron importer and its interaction with other mitochondrial proteins, AbcblO and ferrochelatase, in the acquisition of mitochondrial iron and its utilization in heme synthesis. In an attempt to dentify additional, unknown components important for heme synthesis, we screened thousands of microarrays for genes that were tightly co-expressed and co-regulated with previously known heme biosynthesis genes to identify potentially interesting, novel candidate genes. Follow up studies of these candidate genes in the zebrafish showed that gene-specific knockdown, using anti-sense morpholinos, resulted in profound anemia in all cases. In a complementary approach, we recently analyzed the expression of structural proteins with predicted transmembrane motifs, transporter function, or localization to the mitochondria, which were identified by RNAseq analysis from differentiating fetal liver cells. We identified 9 additional strongly induced genes, whose function in erythroid iron/heme metabolism has not been previously studied. We propose to study the expression and loss-of-function phenotype of these 9 structural genes (c20orf108, Snx3, Slc43a1, Slc43a3, Slc7a5, Ehbplll, Tmcc2, Slc38a5, Tmem14c) in the zebrafish and mammalian cells. In particular, we plan to focus on one of these newly identified genes from the two bioinformatics screens, Tmem14c, a small mitochondrial membrane protein of unknown function. RELEVANCE (See instructions): Elucidating the function of these 8 structural proteins, Tmem14c and its interacting protein partners may give insight into unknown steps in mitochondrial heme metabolism and provide new genetic tools for exploring human disorders of iron/heme metabolism and erythropoiesis.